Cholinesterase Inhibitors for Treating Inflammation

ABSTRACT

A method of treating a subject with a cytokine-mediated inflammatory disorder comprising administering to the subject an effective amount of a pharmaceutically acceptable cholinesterase inhibitor, provided that the inhibitor is not galantamine.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of and claims priority to U.S.patent application Ser. No. 11/893,116, filed Aug. 14, 2007, which is acontinuation of and claims priority to U.S. patent application Ser. No.11/044,649, filed Jan. 27, 2005, now abandoned, which is entitled topriority under 35 U.S.C. §119(e) to U.S. Provisional Application No.60/539,557, filed Jan. 27, 2004 and U.S. Provisional Application No.60/548,461, filed Feb. 27, 2004, all of which applications are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

There is a continuing need for new medications for treating inflammatorydisorders.

SUMMARY OF THE INVENTION

The invention is a method of treating a subject with a cytokine-mediatedinflammatory disorder comprising administering to the subject aneffective amount of a pharmaceutically acceptable cholinesteraseinhibitor. In one embodiment, the pharmaceutically acceptablecholinesterase inhibitor is administered in an amount sufficient toreduce the level of a proinflammatory cytokine. Typically, thecholinesterase inhibitor is not galantamine. More typically, thecholinesterase inhibitor is not galantamine or a derivative thereof.

The invention is also as method reducing proinflammatory cytokinelevel(s) in a subject in need of such reduction, comprisingadministering to the subject an effective amount of a pharmaceuticallyacceptable cholinesterase inhibitor. Typically, the cholinesteraseinhibitor is not galantamine. More typically, the cholinesteraseinhibitor is not galantamine or a derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the reduction in TNF levels (in pg/ml) inmice with sepsis induced by CLP surgery. Throe groups are shown; 1) micetreated with vehicle; 2) mice treated with galantamine at 0.01 mg/kg;and 3) mice treated with galantamine at 0.1 mg/kg.

FIG. 2 is a bar graph showing the reduction in HMGB1 levels (in ng/ml)in mice with sepsis induced by CLP surgery. Three groups are shown: 1)mice treated with vehicle; 2) mice treated with galantamine at 0.01mg/kg; and 3) mice treated with galantamine at 0.1 mg/kg.

FIGS. 3A, 3B and 3C each shows survival percentage of mice with sepsisinduced by CLP surgery. Three groups are shown: 1) mice treated withvehicle (⋄); 2) mice treated with 10 μg/kg galantamine (◯); and 3) micetreated with 100 micrograms/kg galantamine ().

FIG. 4 shows survival percentage of mice with sepsis induced by CLPsurgery. Three groups are shown; 1) mice treated with vehicle (◯); 2)mice treated with 250 μg/kg tacrine (□); and 3) mice treated with 1.25μg/kg huperzine (▴).

FIG. 5 is a bar graph showing galantamine-induced reduction in TNFlevels (in pg/ml) in rats following induction of endotoxemia.

FIG. 6 is a bar plot showing galantamine-induced reduction in TNT levels(in pg/ml) in mice following induction of endotoxemia.

FIGS. 7A-C show survival percentage of mice with lethal endotoxemia thatwere pre-treated with galantamine one hour before endotoxin injection.Four groups of mice are shown; 1) mice treated with vehicle (◯); 2) micetreated with 4 mg/kg galantamine (); 3) mice treated with 2 mg/kggalantamine (▴); and 4) mice treated with 1 mg/kg galantamine (□). FIG.7A shows results for two groups of 10 mice; FIG. 7B shows results forfour groups of 20 mice each; FIG. 7C shows results for four groups ofmice, either 10 or 20 animals as indicated.

FIG. 8A show survival percentage of mice with sepsis induced by cecalligation and puncture that were treated with tacrine post-operatively.Results from three groups of mice are shown: 1) Mice treated with thesaline vehicle (⋄); 2) mice treated with 0.25 μg/kg tacrine (▪); and 3)mice treated with 2.5 μg/kg tacrine ().

FIG. 8B is a bar plot showing the effect of administration of tacrine at0.25 μg/kg and 2.5 μg/kg on serum TNF (pg/ml) in septic mice.

FIG. 8C is a bar plot showing the effect of administration of tacrine at0.25 μg/kg and 2.5 μg/kg on serum HMGB1 (ng/ml) in septic mice.

FIG. 9 shows survival rate of mice with lethal endotoxemia that werepre-treated with galantamine: 1) mice treated with vehicle (◯); and 2)mice treated with 4 mg/kg galantamine ().

FIG. 10 shows survival rate of mice with lethal endotoxemia that werepretreated with Huperzine A: 1) mice treated with vehicle (◯); 2) micetreated with 0.4 mg/kg Huperzine A (); and 3) mice treated with 0.1mg/kg Huperzine A (▴).

FIG. 11 shows survival rate of mice with lethal endotoxemia that werepretreated with Neostigmine: 1) mice treated with vehicle (◯); and 2)mice treated with 0.1 fig/kg Neostigmine ().

FIG. 12 shows survival rate of mice with lethal endotoxemia that werepretreated with Physostygmine: 1) mice treated with vehicle (◯); and 2)mice treated with 0.2 mg/kg Physostigmine ().

DETAILED DESCRIPTION OF THE INVENTION

A “cholinesterase inhibitor” is a compound that inhibits or reduces theactivity of acetylcholinesterase or butyrylcholinesterase. In oneembodiment, the activity of an esterase is reduced by at least 25%. Inanother embodiment, the activity is reduced by at least 50%. In yetanother embodiment, the activity is reduced by least 75%. In anotherembodiment, the activity is reduced by at least 90%. In yet anotherembodiment, the activity is reduced by at least 99%. The activity of acholinesterase is compared to cholinesterase activity in the absence ofthe compound. A “pharmaceutically acceptable” cholinesterase inhibitoris one that does not cause unacceptable side effects in the subjectbeing treated when administered at an effective amount, as the term isdefined herein. Either inhibitors of acetylcholinesterase orbutyrylcholinesterase or dual inhibitors can be used to practice thepresent invention. Acetylcholinesterase inhibitors are preferred.Examples of pharmaceutically acceptable cholinesterase inhibitorsinclude tacrine or tacrine analogues and pharmaceutically acceptablesalts thereof and huperzine A or its analogues and pharmaceuticallyacceptable salts thereof. Any of the following compounds as well astheir analogs and pharmaceutically acceptable salts can be used: Greenmamba snake (Dendroaspis angusticeps) toxin fasciculin, metrifonate,heptyl-physostigmine, norpyridostigmine, norneostigmine, physostigmine,heptylphysostigmine, velnacrine, citicoline, donepezil, metrifonate,7-methoxytacrine, eptastigmine, icopezil, ipidacrine, zifrosilone,anseculin, suronacrine, linopiridine, rivastigmine, physostigmine,neostigmine, edrophonium, demacarium and ambenonium.

As used herein, tacrine refers to a compound of formula (I)

Tacrine and its analogs are described, for example, ion U.S. Pat. Nos.4,562,196, 4,754,050, 4,835,275, 4,839,364, 4,631,286, 4,816,456 and6,194,403, the entire teachings of which are herein incorporated byreference. Tacrine analogs include compounds of formula (II A):

In formula (II A), R₁ represents hydrogen, hydroxy, methyl, methoxy,ethyl or ethoxy; R₁ and R₂ together may form a double bond, R₃ and, R₄together may form a double bond, or R₁, R₂, R₃, and R₄ are all hydrogen;R₅ represents hydrogen, hydroxy, methoxy or ethoxy; R₆ representshydrogen, hydroxy, methoxy or ethoxy; and R₇ represents no radical; anN-oxy radical (N→O); a C1-C20 alkyl radical or a radical selected fromthe group consisting of

wherein each It is independently selected from C1-C20 alkyl; andpharmaceutically acceptable salts thereof. Each of the foregoing alkylgroups may be straight chain or branched.

Tacrine analogs and derivatives are further disclosed in U.S. Pat. Nos.4,562,196, 4,754,050, 4,835,275, 4,839,364, 4,631,286, 4,868,177. Theentire teachings of these are herein incorporated by reference.

Tacrine analogs Miler include compounds of the following formulae:

wherein n is an integer between 2 and 10, X is defined as Cl or F;

wherein n is an integer between 2 and 10, X is defined as H, Cl, F and Yis defined as H, Cl, F but X is not the same as Y;

wherein X is H, Cl, F and Y is H, Cl, F; and

wherein X═H, Cl, F.

Compounds chemically related to tacrine suitable for practicing themethods of the present invention include suronacrine and velnacrine andtheir derivatives:

In formula (II G) n is 1, 2 or 3; X is hydrogen, lower alkyl, loweralkoxy, halogen, hydroxy, nitro, trifluoromethyl, NHCOR2 where R2 isloweralkyl, or NR3R4 where R3 and R4 are independently hydrogen orloweralkyl; R is hydrogen or loweralkyl; R1 is hydrogen, loweralkyl,diloweralkylaminoloweralkyl, arylloweralkyl, diarylloweralkyl,furylloweralkyl, thienylloweralkyl, oxygen-bridged arylloweralkyl,oxygen-bridged diarylloweralkyl, oxygen-bridged furylloweralkyl oroxygen-bridged thienylloweralkyl; Y is C═O or CR5 OH where R5 ishydrogen or loweralkyl; Z is CH2 or C═CR6R7 where R6 and R7 areindependently hydrogen or loweralkyl; or Y and Z taken together isCR5=CH where CR5 and CH correspond to Y and Z respectively; an opticalantipode thereof, or a pharmaceutically acceptable acid addition saltthereof.

As used herein, the term “oxygen-bridged” shall signify the fact that anon-alpha methylene group present in a lower alkyl group (C1-C6)attached to the amino nitrogen which in turn is attached to the lysedring system is replaced by an oxygen atom. Thus, for instance, examplesof oxygen-bridged arylloweralkyl include 3-phenoxypropyl and4-phenoxybutyl, and examples of oxygen-bridged diarylloweralkyl include2-[bis(4-fluorophenyl)methoxy]ethyl and2-[bis(3-fluorophenyl)methoxy)ethyl.

Examples of tacrine analogs suitable for practicing the presentinvention include

In formula (Il H), n is 1, 2 or 3; X is hydrogen, loweralkyl,loweralkoxy, halogen, hydroxy, nitro or trifluoromethyl R1 and R2 areeach independently hydrogen, lower alkyl or arylloweralkyl, but both maynot be arylloweralkyl simultaneously; R3 and R4 are each independentlyhydrogen, lower alkyl, arylloweralkyl, formyl or lower alkylcarbonyl, oralternatively the group —NR3 R4 taken as a whole constitutes:

stereo isomers thereof and pharmaceutically acceptable acid additionsalts thereof.

Additional examples of compounds suitable for practicing the presentinvention are disclosed in U.S. Pat. Nos. 4,550,113, 5,397,785,5,536,728, 5,861,411 and 6,433,173, the entire teachings of which areincorporated herein by reference. Examples of these compounds includeipidacrine:

Derivatives of ipidacrine suitable for practicing the present inventioninclude:

In formula (III A), wherein R1 represents a C2-C6 alkyl group or a grouprepresented by the following formula;

wherein R2 and R3 together with the nitrogen atom to which both R2 andR3 are attached represent

and n represents 0 or an integer from 1 to 3; and further wherein eachof R10 and R11 independently represents a hydrogen atom or a C1-C4 alkylgroup; and a pharmaceutically acceptable acid addition salt thereof.

Additional derivatives of ipidacrine include;

In formula (III B), R1 represents a group represented by the followingformula:

wherein wherein each of R2 and R3 independently represents a hydrogenatom, C1-C6 alkyl group, C3-C6 cycloalkyl group or

wherein each of R4 and R5 independently represents a hydrogen atom orC1-C6 alkyl group, and n represents 0 or an integer from 1 to 3;or wherein R2 and R3 together with the nitrogen atom to which both R2and R3 are attached represent

wherein R6 represents a hydrogen atom or C1-C6 alkyl group, and nrepresents 0 or an integer from 1 to 3; ring A represents any of thefollowing:

wherein R7 represents a hydrogen atom, C1-C6 alkyl group or halogenatom, R8 and R9 independently represents a hydrogen atom or C1-C4 alkylgroup, R10 and R11 independently represents a hydrogen atom or C1-C4alkyl group; and ring B represents any of the following:

wherein each of R12 and R13 independently represents a hydrogen atom orC1-C4 alkyl group or R12 and R13 may be combined together to form aC2-C6 alkylene group,

As used herein, huperzine A is a compound of formula (IV):

Huperzine A and its analogs are described, for example, in U.S. Pat.Nos. 5,104,880 and 5,929,084, the entire teachings of both of which areherein incorporated by reference.

Huperzine A analogs include compounds represented by formula (IV):

In formula (IV), R1 is H, (C1-C8)alkyl or halo; R2 is H or (C1-C8)alkyl;R3 and R4 are individually H, (C1-C)alkyl, NO₂, hydroxy or halo; R5 andR6 are individually H,(C1-C8)alkyl, aryl or aralkyl; R7 is 1-1, halo or(C1-C8)alkyl, R8 is halo or (C1-C8)alkyl, R9 is absent or is H; and thebonds represented by—are individually absent or, together with theadjacent bond, form the unit C═C, with the proviso that if both of thebonds represented by—are present, R3 and 114 cannot both be H unless R7or R8 is halo.

The disclosed method in one aspect excludes the use of huperzine Aand/or a derivative thereof. In another aspect, huperzine A (and/or aderivative thereof) and galantamine (and/or a derivative thereof) areexcluded.

As used herein, galantamine is represented by structure (V):

Galantamine derivatives excluded from practice of the method includemetabolites of galantamine such as those shown below:

As used herein physostigmine is represented by formula (VII):

Derivatives of physostigmine are disclosed for example in U.S. Pat. Nos.4,831,155, 4,914,102, 4,978,155, 5,081,117, 5,306,825, disclosures ofwhich are incorporated herein by reference in their entirety. Examplesof derivatives suitable for practicing the methods of the presentinvention include:

where R and R′ are independently selected from the group consisting ofpropyl, isopropyl, tert-butyl, phenyl, cyclohexyl, heptyl, undecyl andpentadecyl or R and R′ are both ethyl or methyl.

Derivatives of physostigmine further include:

where X is hydrogen, halogen or loweralkyl; andR1, R2 and R3 are each independently hydrogen, loweralkyl, cycloalkyl,arylloweralkyl or aryl, or alternatively the group —NR2 R3 taken as awhole constitutes

R4 being hydrogen or lower alkyl. In the above definition, the term“lower” means the group it is describing contains from 1 to 6 carbonatoms,

Derivatives of physostigmine further include:

where R1 is alkyl, cycloalkyl, bicyoloalkyl, aryl or arylloweralkyl; R2is hydrogen or alkyl or the group —NR1R2 taken together forms amonocyclic or bicyclic ring of 5 to 12 carbons; m is 0, 1 or 2; each Xis independently hydrogen, halogen, loweralkyl, nitro or amino; the term“arylloweralkyl” signifies a monovalent substituent which consists of an“aryl” group as defined by the formula

where n is an integer of 1 to 3 and Z is hydrogen, halogen, loweralkyl,loweralkoxy, trifluoromethyl, nitrogen and amino, linked through aloweralkylene group; and the term “bicyoloalkyl” signifies abicycloalkyl group having from 7 to 11 carbon atoms; or the opticalisomers including the 3aS-cis and 3aR-cis optical isomers, or a racemicmixture or a pharmaceutically acceptable acid addition salt thereof.

As used herein, rivastigmine is represented by formula

Examples of suitable derivatives of rivastigmine are disclosed in U.S.Pat. No. 4,948,807, the entire teachings of which are incorporatedherein by reference.

Derivatives of rivastigmine include compounds of formula;

wherein R1 is hydrogen, C1-C6 alkyl, cyclohexyl, allyl or benzyl, R2 ishydrogen, methyl, ethyl or propyl, or R1 and R2 together with thenitrogen to which they are attached form a morpholino or piperidinoradical, R3 is hydrogen or lower alkyl, R4 and R5 are the same ordifferent and each is a lower alkyl, and the dialkylaminoalkyl group isin the meta, ortho or para position, or a pharmacologically acceptablesalt thereof.

As used herein, the terms piperidino and morpholino radicals refer tothe following structures:

Donepezil, as used herein, is represented by formula (IX):

Derivatives of donepezil suitable for practicing the present inventionare described, for example, in U.S. Pat. Nos. 4,895,841 and 5,100,901,the entire teachings of which are incorporated herein by reference.Examples of donepezil derivatives include compounds of formula (IX A):

In formula (IX A), r is an integer of 1 to 10, R22 is hydrogen ormethyl, and the R22 radicals can be the same or different when r is from2 to 10; K is phenylalkyl or phenylalkyl having a substituent on thephenyl ring; S is hydrogen or a substituent on the phenyl ring, and t isan integer of 1 to 4, with the proviso that (S)t can be a methylenedloxygroup or an ethylenedloxy group joined to two adjacent carbon atoms ofthe phenyl ring; and q is an integer of 1 to 3.

Derivatives of donepezil further include compounds of formula (IX B):

In formula (IX B), J is

-   (a) a group, substituted or unsubstituted, selected from the group    consisting of (1) phenyl, (2) pyridyl, (3) pyrazyl, (4)    quinolyl, (5) cyclohexyl, (6) quinoxalyl and (7), furyl;-   (b) a monovalent or divalent group, in which the phenyl may have a    substituent(s), selected from the group consisting of (1)    indanyl, (2) indanonyl, (3) indenyl, (4) indenonyl, (5)    indanedlonyl, (6) tetralonyl, (7) benzosuberonyl, (8) indanolyl    and (9) C6H5-CO—CH(CH3)-;-   (c) a monovalent group derived from a cyclic amide compound;-   (d) a lower alkyl or-   (e) a group of R21-CH═CH— in which R21 is hydrogen or a lower    alkoxycarbonyl;-   B is —(CHR22)r-, —CO—(CHR22)r-, —NR4-(CHR22)r-, R4 being hydrogen, a    lower alkyl, an acyl, a lower alkylsulfonyl, phenyl, a substituted    phenyl, benzyl or a substituted benzyl, —CO—NR5-(CHR22)r-, R5 being    hydrogen, a lower alkyl or phenyl, —CH═CH—(CHR22)r-, —OCOO—(CHR22)r,    —OOC—NH—(CHR22)r-, —NH—CO—(CHR22)r-, —CH2-CO—NH—(CHR22)r-,    —(CH2)2-CO—NH—(CHR22)r-, —CH(OH)—(CHR22)r-, r being zero or an    integer of 1 to 10, R22 being hydrogen or methyl so that one    alkylene group may have no methyl branch or one or more methyl    branch, ═(CH—CH═CH)b, b being an integer of 1 to 3, ═CH—(CH2)c-, c    being zero or an integer of 1 to 9, ═(CH—CH)d=, d being zero or an    integer of 1 to 5; —CO—CR═CH—CH2-, —CO—CH2-CH(OH)—CH2-,    —CH(CH3)-CO—NH—CH2-, —CH═CH—CO—N—(CH2)2-, —NH—, —O—, —S—, a    dialkylaminoalkylcarbonyl or a lower alkoxycarbonyl; T is nitrogen    or carbon; Q is nitrogen, carbon or —N→O; and q is an integer of 1    to 3;-   K is hydrogen, phenyl, a substituted phenyl, an arylalkyl in which    the phenyl may have a substituent, cinnamyl, a lower alkyl,    pyridylmethyl, a cycloalkylalkyl, adamantanemethyl, furylmethyl, a    cycloalkyl, a lower alkoxycarbonyl or an acyl; and shows a single    bond or a double bond.

Zifrosilone, as used herein, is represented by formula (X):

Derivatives of zifrosilone are disclosed, for example, in U.S. Pat. Nos.5,693,668, 5,554,780, 5,760,267, the entire teachings of which areincorporated herein by reference. Examples of zifrosilone derivativesinclude:

In formula (X A), Z is —C(O)C(O)W, —C(O)CF2 CF3, or —(CH2)n-Q-CF2X, eachof m and n is zero or one with the proviso that the sum of m and n isless than two, Q is —C(O)—, or —CHOC(O)R with R being H or C1-C10 alkylX is X′ or X″ with X′ being H, Br, Cl, P or R.sub.4 and X″ being COR9,CO2R5, CONHR5 or COR6, R1, R2, R3 and R4 each being C1-C10 alkyl, or(CH2)p aryl, with p being zero, one or two, R′ or R5 are each H, C1-C10alkyl, phenyl, benzyl or phenethyl, R9 is C1-C10 alkyl, phenyl, benzylor phenethyl, R6 is (NHCHR7 C(O))qR8 with R7 being the residue of anynatural occurring alpha-amino acid, q is one to four and R8 is —OR5 orNHR5, Y is H, OH, (C1-6) alkyl, (C1-6) alkoxy, hydroxy (C1-6) alkyl,amino(C1-6) alkyl, NH2, azido, CN, CO2 R5, COR9, —SO3 H, Br, Cl, F or—(CH2)xSiR1R2R3 with x being zero, one or two.

In other embodiments, the following compounds can be used to practicethe methods of the present invention: Arecoline, represented by formula(XI), Xanomeline, represented by formula (XII), Subcomeline, representedby formula (XIII), Cevimeilne, represented by formula (XIV), Alvameline,represented by formula (XV), Milameline, resented by formula (XVI), orTalsacildine, presented by formula (XVII):

In other embodiments, compounds of formulae (XVIII)-(XXI) can be used topractice the methods of the present invention:

Compounds of formula (XI) through (XXI) are disclosed, for example, inGreenlee et al., Il Farmaco 56 (2001): 247-250, the entire teachings ofwhich is incorporated herein by reference.

The term “alkyl”, as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight or branchedmoieties, typically C₁-C₁₀, preferably C₁-C₆. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl, andt-butyl.

As used herein, the term “lower alkyl” refers to a C1-C6

The term “alkenyl”, as used herein, unless otherwise indicated, includesalkyl moieties having at least one carbon-carbon double bond whereinalkyl is as defined above. Examples of alkenyl include, but are notlimited to, ethenyl and propenyl.

The term “alkynyl”, as used herein, unless otherwise indicated, includesalkyl moieties having at least one carbon-carbon triple bond whereinalkyl is as defined above. Examples of alkynyl groups include, but arenot limited to, ethynyl and 2-propynyl.

The terms “alkoxy”, as used herein, means an “alkyl-O-” group, whereinalkyl is as defined above.

The term “cycloalkyl”, as used herein, unless otherwise Indicated,includes non-aromatic saturated cyclic alkyl moieties wherein alkyl isas defined above. Examples of cycloalkyl include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl,“Bicycloalkyl” groups are non-aromatic saturated carbocyclic groupsconsisting of two rings. Examples of bicycloalkyl groups include, butare not limited to, bicyclo-[2.2.2]-octyl and norbornyl. The term“cycloalkenyl” and “bicycloalkenyl” refer to non-aromatic carbocycliccycloalkyl and bicycloalkyl moieties as defined above, except comprisingof one or more carbon-carbon double bonds connecting carbon ring members(an “endocyclic” double bond) and/or one or more carbon-carbon doublebonds connecting a carbon ring member and an adjacent non-ring carbon(an “exocyclic” double bond). Examples of cycloalkenyl groups include,but are not limited to, cyclopentenyl and cyclohexenyl. A non-limitingexample of a bicycloalkenyl group is norborenyl. Cycloalkyl,cycloalkenyl, bicycloalkyl, and bicycloalkenyl groups also includegroups similar to those described above for each of these respectivecategories, but which are substituted with one or more oxo moieties.Examples of such groups with oxo moieties include, but are not limitedto oxocyclopentyl, oxocyclobutyl, oxocyclopentenyl, and norcamphoryl.

The term “cycloalkoxy”, as used herein, unless otherwise indicated,includes “cycloalkyl-O-” group, wherein cycloalkyl is defined above.

The term “aryl”, as used herein, refers to carbocyclic group. Examplesof aryl groups include, but are not limited to phenyl and naphthyl.

The term “heteroaryl”, as used herein, refers to aromatic groupscontaining one or more heteroatoms (O, S, or N). A heteroaryl group canbe monocycle or polycyclic. The heteroaryl groups of this Invention canalso include ring systems substituted with one or more oxo moieties.Examples of heteroaryl groups include, but are not limited to,pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, furyl, thienyl,isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnollnyl,indazolyl, indollzinyl, phihalazinyl, pyridazinyl, triazinyl,isoindolyl, purinyl, oxadlazolyl, thiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzotriazolyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxallnyl, naphthyridinyl,dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl,tetrahydroisoquinolyl, benzofuryl, fluropyridinyl, pyrolopyrimidinyl,and azaindolyl.

The foregoing heteroaryl groups may be C-attached or N-attached (wheresuch is possible). For instance, a group derived from pyrrole may bepyrrol-1-yl(N-attached) or pyrrol-3-yl(C-attached).

In the context of the present invention, a bicyclic carbocyclic group isa bicyclic compound holding carbon only as a ring atom. The ringstructure may in particular be aromatic, saturated, or partiallysaturated. Examples of such compounds include, but are not limited to,indanyl, naphthalenyl, azulenyl.

In the context of the present invention, an amino group may be a primary(—NH₂), secondary (—NHR_(a)), or tertiary (—NR_(a)R_(b)), wherein R_(a)and R_(b) may be any of the alkenyl, alkynyl, alkoxy, cycloalkyl,cycloalkoxy, aryl, heteroaryl, and a bicyclic carbocyclic group.

As used herein, a cytokine is a soluble protein or peptide which isnaturally produced by mammalian cells and which act in vivo as humeralregulators at micro to picomolar concentrations. Cytokines can, eitherunder normal or pathological conditions, modulate the functionalactivities of individual cells and tissues. A proinflammatory cytokineis a cytokine that is capable of causing any of the followingphysiological reactions associated with inflammation: vasodialation,hyperemia, increased permeability of vessels with associated edema,accumulation of granulocytes and mononuclear phagocytes, or depositionof fibrin. In some cases, the proinflammatory cytokine can also causeapoptosis, such as in chronic heart failure, where TNF has been shown tostimulate cardlomyocyte apoptosis (Pulkki, 1997; Tsutsul et al., 2000).Nonlimiting examples of proinflammatory cytokines are tumor necrosisfactor (TNF), interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-18, interferonγ, HMGB1 (see U.S. Pat. Nos, 6,468,533 and 6,448,223, the entireteachings of which are incorporated herein by reference),platelet-activating factor (PAF), and macrophage migration inhibitoryfactor (MIF).

A “subject in need of reduction of pro-inflammatory cytokine levels” isa subject with a disease or condition caused by levels of one or morepro-inflammatory cytokines that are higher than normal in one or moreregions of the subject's body. Examples of such conditions include theinflammatory disorders discussed below.

An “amount sufficient to reduce the level of a proinflammatory cytokine”is an amount sufficient to decrease the level of one or moreproinflammatory cytokine in a sample, for example, in a blood dr serumsample. When referring to the effect of the cholinesterase inhibitor onrelease of proinflammatory cytokines, the use of the terms “inhibit” or“decrease” encompasses at least a small but measurable reduction inproinflammatory cytokine release. In one embodiment, the release of theproinflammatory cytokine is inhibited by at least 20% over non-treatedcontrols; in another embodiment, the inhibition is at least 50%; inanother embodiment, the inhibition is at least 70%, and in yet anotherembodiment, the inhibition is at least 80%, In one embodiment, thecholinesterase inhibitor is administered in an amount sufficient todecrease the level of a proinflammatory cytokine selected from the groupconsisting of TNF and HMGB1. Levels of pro-Inflammatory cytokines in asample can be determined by any of the method known in the art, such as,for example, an immunosorbtion assay.

Examples of cytokine-mediated inflammatory disorders which can betreated using the present invention include appendicitis, peptic,gastric or duodenal ulcers, ileus, peritonitis, pancreatitis,diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis,hepatitis, enteritis, Whipple's disease, asthma, allergy, anaphylacticshock, organ ischemia, reperfusion injury, organ necrosis, hay fever,sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia,eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion,epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema,rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscoplosilicovolcanoconiosis, alveolitis, bronchiolltis, pharyngitis, pleurisy,sinusitis, influenza, respiratory syncytial virus, herpes, disseminatedbacteremia, Dengue fever, candldiasis, malaria, filariasis, amebiasis,hydatld cysts, vasulitis, angiltis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocardial ischemia, periarteritisnodosa, coellac disease, congestive heart failure, adult respiratorydistress syndrome, meningitis, encephalitis, cerebral infarction,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinalcord injury, paralysis, uveitis, osteomyelitis, fasciitis, Paget'sdisease, gout, periodontal disease, synovitis, thryolditis,Goodpasture's syndrome, Behcet's syndrome, allograft rejection,graft-versus-host disease, Type I diabetes, Berger's disease, Type IIdiabetes, Berger's disease, Retier's syndrome, or Hodgkins disease. Forpurposes of the disclosed invention, Alzheimer's disease is notconsidered to be an “inflammatory disease”.

The disclosed method is particularly useful for treating appendicitis,peptic, gastric or duodenal ulcers, peritonitis, pancreatitis,hepatitis, asthma, allergy, anaphylactic shock, organ ischemia,reperfusion injury, organ necrosis, hay fever, sepsis, septicemia,endotoxic shock, cachexia, septic abortion, disseminated bacteremia,congestive heart failure, adult respiratory distress syndrome, cerebralinfarction, cerebral embolism, sepsis, spinal cord injury, paralysis,allograft rejection or graft-versus-host disease.

In another embodiment, the disclosed method is particularly useful fortreating appendicitis, peptic, gastric or duodenal ulcers, peritonitis,pancreatitis, hepatitis, asthma, allergy, anaphylactic shock,reperfusion injury, organ necrosis, hay fever, septicemia, endotoxicshack, cachexia, septic abortion, disseminated bacteremia, burns, adultrespiratory distress syndrome, cerebral infarction, cerebral embolism,sepsis, spinal cord injury, paralysis, allograft rejection orgraft-versus-host disease.

The disclosed method is also particularly effective in treatingappendioitis, sepsis, peritonitis, pancreatitis, organ ischemia,reperfusion injury, endotoxic shock, cachexia, adult respiratorydistress syndrome, chronic obstructive pulmonary disease, myocardialischemia, allograft rejection, congestive heart failure, cystic fibrosisand graft-versus-host disease,

In another embodiment, the disclosed method is particularly effective intreating asthma, peritonitis, pancreatitis, reperfusion injury,endotoxic shock, cachexia, adult respiratory distress syndrome,allograft rejection, cystic fibrosis and graft-versus-host disease.

In yet another embodiment, the present invention is effective intreatment of aspects of sepsis.

As used herein, a “subject” includes mammals, e,g., humans, companionanimals (e.g., dogs, cats, birds and the like), farm animals (e.g.,cows, sheep, pigs, horses, fowl and the like) and laboratory animals(e.g., rats, mice, guinea pigs and the like). In a preferred embodimentof the disclosed methods, the subject is human.

Cholinesterase inhibitors are typically administered as a pharmaceuticalcomposition that comprises (or consists of) the cholinesterase inhibitorthat is greater than 95% and preferably greater than 99% pure by weightand one or more exoipients, diluents or other inert ingredients commonlyfound in pharmaceutical compositions. Thus, any cholinesterase inhibitorthat are natural products, i.e., produced in nature, are isolated andpurified or produced synthetically before being used in the disclosedmethod.

As used herein, an “effective amount” of a compound of the disclosedinvention is a quantity which, when administered to a subject in need oftreatment, improves the prognosis of the subject, e.g., delays the onsetof and/or reduces the severity of one or more of the subject's symptomsassociated with condition being treated. The amount of thecholinesterase inhibitor to be administered to a subject will depend onthe particular disease, the mode of administration, the bioavailabilityof the cholinesterase inhibitor and the characteristics of the subject,such as general health, other diseases, age, sex, genotype, body weightand tolerance to drugs. The skilled artisan will be able to determineappropriate dosages depending on these and other factors. Effectiveamounts of a pharmaceutically acceptable cholinesterase inhibitortypically ranges between about 0.1 mg/kg body weight per day and about1000 mg/kg body weight per day, and preferably between 1 mg/kg bodyweight per day and 100 mg/kg body weight per day.

The route of administration of the cholinesterase inhibitor depends onthe condition to be treated. For example, intravenous injection may bepreferred for treatment of a systemic disorder such as septic shock, andoral administration may be preferred to treat a gastrointestinaldisorder such as a gastric ulcer. The route of administration and thedosage of the cholinesterase inhibitor to be administered can bedetermined by the skilled artisan without undue experimentation inconjunction with standard dose-response studies. Relevant circumstancesto be considered in making those determinations include the condition orconditions to be treated, the choice of composition to be administered,the ago, weight, and response of the individual patient, and theseverity of the patient's symptoms. Thus, depending on the condition,the cholinesterase inhibitor can be administered orally, parenterally,intranasally, vaginally, rectally, lingually, sublingually, bucally, andintrabuccaly to the patient.

Accordingly, cholinesterase inhibitor compositions designed for oral,lingual, sublingual, buccal and intrabuccal administration can be madewithout undue experimentation by means well known in the art, forexample with an inert diluent or with an edible carrier. Thecompositions may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, thepharmaceutical compositions of the present invention may be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragaoanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

Cholinesterase inhibitor compositions of the present Invention caneasily be administered parenterally such as for example, by intravenous,intramuscular, intrathecal or subcutaneous injection. Parenteraladministration can be accomplished by incorporating the cholinergicagonist compositions of the present invention into a solution orsuspension. Such solutions or suspensions may also include sterilediluents such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other synthetic,solvents. Parenteral formulations may also include antibacterial agentssuch as for example, benzyl alcohol or methyl parabens, antioxidantssuch as for example, ascorbic acid or sodium bisulfite and chelatingagents such as EDTA, suffers such as acetates, citrates or phosphatesand agents for the adjustment of tonicity such as sodium chloride ordextrose may also be added. The parenteral preparation can be enclosedin ampules, disposable syringes or multiple dose vials made of glass orplastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the cholinesterase inhibitor in the glycerin, mixing theheated glycerin after which purified water may be added, and pouring thehot mixture into a suppository mold.

The present invention includes nasally administering to the subject aneffective amount of the cholinesterase inhibitor. As used herein,nasally administering or nasal administration includes administering thecholinesterase inhibitor to the mucous membranes of the nasal passage ornasal cavity of the patient. As used herein, pharmaceutical compositionsfor nasal administration of a cholinesterase inhibitor includetherapeutically effective amounts of the cholinesterase inhibitorprepared by well-known methods to be administered, for example, as anasal spray, nasal drop, suspension, gel, ointment, cream or powder.Administration of the cholinesterase inhibitor may also take place usinga nasal tampon or nasal sponge.

The cholinesterase inhibitor can be administered alone (as amonotherapy) or in combination with one or more other pharmaceuticallyactive agents that are effective against the condition being treated.However, the combination therapy does not include a choline esterasereactivator, as that the term is used in U.S. Pat. No. 5,981,549, theentire teachings of which are incorporated herein by reference. Forexample, they can be administered in combination with an acetylcholinereceptor agonist (particularly alpha 7 specific agonists and muscarinicreceptor agonists that penetrate the blood brain barrier, see, forexample, U.S. Pat. No. 6,610,713 and WO 03/072135 and U.S. Ser. No.10/729,427, filed Dec. 5, 2003—the entire teachings of these threepublications are incorporated herein by reference), e.g.,anti-microblals, anti-inflammatory agents, analgesics, anti-viralagents, anti-fungals, anti-histamines and the like. Cholinesteraseinhibitors are particularly effective when administered in combinationwith other anti-inflammatory agents and with alpha7 specific cholinereceptor agonists.

Examples of suitable anti-inflammatory agents include examples ofsuitable NSAIDs include aminopolycarboxylic acid derivatives (e.g.,Enfenamic Acid, Etofenamate, Flufenamie Acid, Isonixin, MeclofentunicAcid, Niflurnic Acid, Talniflumate, Terofenamato and Tolfenatnic Acid),arylacetic acid derivatives (e.g., Acematlein, Alclofenac, Amfenac,Bufexamac, Caprofen, Cinmetacin, Clopirao, Diclofenac, DiclofenacSodium, Etodolac, Felbinac, Fenclofenac, Fenclorao, Fenolozic Acid,Fenoprofen, Fentiazac, Flutbiprofen, Olueatnetacin, Ibufenac, Ibuprofen,Indomethacin, Isofezolac, Isoxepac, Ketoprofen, Lonazolao, MetiazinicAcid, Naproxen, Oxametacine, Proglumrtacin, Sulindac, Tenidap, Tiramide,Tolectin, Tolmetin, Zomax and Zomepirac), arylbutyric acid ferivatives(e.g., Burnadizon, Butibufen, Fenbufen and Xenbucin) arylcarboxylicacids (e.g., Clidanac, Ketorolac and Tinorldine), arylproprionlc acidderivatives (e.g., Almlnoprofen, Benoxaprofen, Buoloxic Acid, Carprofen,Fenoprofen, Flunoxaprofen, Flutbiprofen, Ibuprofen, Ibuproxam,Indoprofen, Ketoprofen, Loxoprofen, Miroprofen, Naproxen, Oxaprozin,Pikotoprofen, Piroprofen, Pranoprofen, Protinizinic Acid, Suprofen andTiaprofenic Acid), pyrazoles (e.g., Difenamizole and Epirizole),pyrazolones (e.g., Apazone, Benzpiperylon, Feprazone, Mofebutazone,Morazone, Oxyphenbutazone, Phenylbutazone, Plpebuzone, Propyphenazone,Ramifonazone, Suxibuzone and Thiazolinobutazone), salleyollc acidderivatives (e.g., Acetaminosniol, 5-Arninosalicylic Acid, Aspirin,Benorylate, Biphenyl Aspirin, Brornosaligenin, Calcium Acetylsalicylate,Diflunisal, Etersalate, Fendosal, Flufenisal, Gentisic Acid, GlycolSalicylate, Imidazole Salicylate, Lysine Acetylsalicylate, Meselamine,Morpholine Salicylate, 1-Naphthyl Sallicylate, Olsalazine, Parsaimide,Phenyl Acetylsalicylate, Phenyl Salicylate, 2-Phosphonoxybenzoic Acid,Salacetamide, Sallcylamide O-Acetic Acid, Salicylic Acid, SalicyloylSalicylic Acid, Salicylsulfuric Acid, Salsalate and Sulfasalazine),thlazinecarboxamides (e.g., Droxicam, Isoxicam, Piroxicam andTenoxicam), ε-Aeetamidoceprole Acid, S-Adenosylinethionine,3-Amino-4-hydroxybutyric Acid, Amixetrine, Bendazac, Benzydamine,Biwatome, Difenpiramidc, Ditazol, Emorfazone, Guaiazulene, Ketorolac,Meelofenamic Acid, Mefenamic Acid, Nabumetone, Nimesulide, Orgotein,Oxaeeprol, Paranyline, Perisoxal, Pifoxime, Piroxicam, Proquazone,Tenidap and a COX-2 inhibitor (e.g., Rofecoxib, Valdecoxib andCelecoxlb).

The invention is illustrated by the following examples which are notintended to be limiting in any way.

Exemplification EXAMPLE 1 Galantamine Treatment Reduces Serum TNF Levelsin Septic Mice

Cecal Ligation and Puncture (CLP) was performed as described in Fink andHeard, J. of Surg. Res, 49:186-196 (1990), Wichman et al., Crit, CareMed. 26:2078-2086 (1998) and Remick et al., Shock 4:89-95 (1995).Briefly, BALB/c mice were anesthetized with 100 mg/kg ketamine (FortDodge, Fort Dodge, Iowa) and 10 mg/kg of xylazine (Bohringer Ingelhelm,St. Joseph, Mo.) intramuscularly. A midline incision was performed, andthe mum was Isolated. A 6-0 prolene suture ligature was placed at alevel 5.0 mm from the cent tip away from the ileocecal valve.

The ligated cecal stump was then punctured once with a 22-gauge needle,and 2 mm stool extruded. The cecum was then placed back into its normalintra-abdominal position. The abdomen was then closed with a runningsuture of 6-0 prolene in two layers, peritoneum and fascia separately toprevent leakage of fluid. All animals were resuscitated with a normalsaline solution administered sub-cutaneously at 20 ml/kg of body weight.Each mouse received a subcutaneous injection of imipenem (0.5 mg/mouse)(Primaxin), Merck & Co., Inc., West Point, Pa.) 30 minutes after thesurgery. Animals were then allowed to recuperate.

Twenty-four hours after surgery, mice were intraperitoneallyadministered either 0.01 mg/kg or 0.1 mg/kg Galantamine Hydrobromide(CalBioChem, San Diego, Calif.) or vehicle control (saline). Galantaminewas prepared in saline to a final concentration of 10 mg/ml. Mice wereeuthanized 6 hours after treatment with Galantamine or vehicle control(30 hours after surgery) and blood was collected by cardiac puncture forTNF-α measurement, TNP-α was measured by ELISA (mouse ELISA kit from R&DSystems Inc., Minneapolis, Minn.).

As shown in FIG. 1, treatment with 0.01 and 0.1 mg/kg Galantamine aftercecal ligation and puncture significantly decreased the serum levelTNF-α by approximately 50 and 80%, respectively, compared to micetreated with vehicle control.

EXAMPLE 2 Galantamine Treatment Reduces Serum HMGB1 Levels in SepticMice

Cecal Ligation and Puncture (CLP) was performed as described above inExample 1. Twenty-four hours after surgery, mice were intraperitoneallyadministered either 0.01 mg/kg or 0.1 mg/kg Galantamine or vehiclecontrol (saline). Mice were euthanized 6 hours after treatment withGalantamine or vehicle control (30 hours after surgery) and blood wascollected by cardiac) puncture for HMGB1 measurement. HMGB1 was measuredby western blot using anti-HMG1 polyclonal antisera (U.S. Pat. No.6,303,321) and standard methods. Band densities were measured using aBio-Rad Imaging densitometer. The results are presented in FIG. 2 whichcompares serum HMGB1 concentration (ng/ml) in Galantamine and vehiclecontrol treated mice.

As shown in FIG. 2, treatment with 0.1 mg/kg Galantamine after cecalligation and puncture decreased the serum level of HMGB1 by about 50%compared to mice treated with vehicle control.

EXAMPLE 3 Galantamine is Protective in Murine CLP Model of Sepsis

Cecal Ligation and Puncture (CLP) was performed as described above inExample 1, Twenty-fours after CLP, mice were treated with either 10ug/kg or 100 us/kg Galantamine or with saline (vehicle control).Galantamine and vehicle control were administered intraperitoneally(i.p.) twice a day for three consecutive days beginning 24 hours aftersurgery (at 24, 30, 48, 54, 72 and 78 hours post-surgery). Mortality wasmonitored twice daily for 14 days after surgery. The results arepresented in FIG. 3, which shows the percentage of surviving animalsfollowing treatment with 10 ug/kg Galantamine, 100 ug,/kg Galantamine orvehicle control. FIG. 3A shows the results of one experiment in which 8mice were treated with 100 ug/kg Galantamine, 8 mice were treated with10 ug/kg Galantamine and 9 mice were treated with vehicle control. FIG.3B shows the results of a second experiment in which 11 mice weretreated with 100 ug/kg Galantamine, 11 mice were treated with 10 us/kgGalantamine and 12 mice were treated with vehicle control. FIG. 3C showsthe results of the two experiments combined; a total of 19 mice weretreated with 100 ug/kg Galantamine, 19 mice were treated with 10 ug/kgGalantamine and 20 mice were treated with vehicle control.

As shown in FIG. 1C, on Day 6, 90% of the mice treated with 100 ug/kgGalantamine survived and 58% of the mice treated with 10 ug/kgGalantamine, whereas only 37% of the mice treated with the vehiclecontrol had survived. These results demonstrate that a dose of 100 ug/kgGalantamine improved survival in the murine CLP model of sepsis.

EXAMPLE 4 Tacrine and Huperzine A are Protective in Murine CUP Model ofSepsis

Cecal Ligation and Puncture (CLP) was performed as described above inExample 1. Twenty-fours after CUP, mice were treated with either 250ug/kg Tacrine (Sigma Chemical Co., St. Louis, Mo.) (n=12), 1.25 ug/kgHuperzine A (Sigma Chemical Co., St. Louis, Mo.) (n=12) or with vehiclecontrol of saline (n=14). Tacrine was prepared in saline and Huperzine Awas suspended in methanol then diluted into saline. Tacrine, Huperzine Aand vehicle control were administered intraperitoneally (i.p.) twice aday for three consecutive days beginning about 24 hours after surgery(at 24, 48 and 72 hours post-surgery). Mortality was monitored twicedaily for 6 days after surgery. The results are presented in FIG. 4,which shows the percentage of surviving animals following treatment withTacrine, Huperzine A or vehicle control.

As shown in FIG. 4, on Day 6, all of the mice treated with 250 ug/kgTacrine survived and about 80% of mice treated with Huperzine survivedwhereas only about 50% of the mice treated with the vehicle control hadsurvived. These results demonstrate that the acetylcholinesteraseinhibitors, Tacrine and Huperzine A, significantly improved survival inthe murine CLP model of sepsis.

EXAMPLE 5 Acetylcholinesterase Inhibitors Galantamine and TacrineImprove Survival in a Murine Model of Sepsis

It has recently been shown that central administration of theacetylcholinesterase inhibitor galantamine attenuates serum TNF levelsduring endotoxemia. Peripheral administration of galantamine, whichcrosses the blood brain barrier, also causes firing of the vagus nerve.The goal of this study was to test the therapeutic efficacy ofgalantamine and tacrine (another centrally-acting acetylcholinesteraseinhibitor) in the mat ligation and puncture (CLP) model of sepsis. Micewere subjected to CLP and treated intraperitoneally with drug orvehicle, twice daily, for 3 consecutive days, beginning 24 h aftersurgery; survival was monitored for 3 weeks. Galantamine significantlyand dose-dependently increased survival from lethal sepsis(vehicle-treated survival=37%; vs. galantamine [10 μg/kg] survival=58%;vs. galantamine [100 μg/kg) survival=90%, p<0.05). Similarly, tacrinesignificantly protected mica from lethal sepsis (vehicle-treatedsurvival=50%; vs. tacrine [250μg/kg] survival=100%, p<0.05). Theseresults indicate that activation of central cholinergic pathwayscontributes to protection against sepsis, Acetyloholinestoraseinhibitors may be novel anti-inflammatory therapeutics by activating theefferent part of the inflammatory reflex.

EXAMPLE 6 Galantamine Inhibits Circulating TNF During Endotoxemia

Endotoxemia was induced in rats via Intraperitoneal (IP) injection ofendotoxin (lipopolysaccharide, GPS; E. coli 0111:B4, Sigma; 10 mg/ml inPBS) (10-30mg/kg, IP). Rats were anesthetized with ketamine (10%) inxylazine and placed in a stereotatic head frame (Stoelting Co.). Theincisor bar was adjusted until the plane defined by the lambda andbregma was parallel to the base plate. The needle of a Hamilton syringe(25 ml) was stereotactically guided into lateral ventricle (0.8 mmposterior to bregma, 1.5 mm lateral to midline, 3.5 mm below the dura).Galantamine at 1, 10, 100 and 1000 ng/kg was dissolved in sterileendotoxin-free saline and administered over 2 min. Vehicle control(saline) was also administered over 2 min. The location ofintracerebroventelcular (i.c.v.) injections was confirmed byhistological examination of the brain after the experiment. TNF-α wasmeasured as described above.

As shown in FIG. 5, treatment with Galantamine (ICV) 1 hour beforeendotoxemia significantly decreased the serum level TNF-α byapproximately 94% compared to mice treated with vehicle control(saline).

EXAMPLE 7 Galantamine Inhibits Serum TNF in Endotoxemia Mice

Male BALB/c mice were injected intraperitoneally with 6 mg/kg endotoxin(lipopolysaccharide, E. coli 0111:84, Sigma, St. Louis, Mo.; 10 mg/ml inpyrogen-free saline), which had been sonicated for 30 min. Galantamine(CalBioChem, San Diego, Calif.) at doses of 0.1, 1, 2 or 4 mg/kg orvehicle control were injected intraperitoneally one hour beforeendotoxin administration in anesthetized animals. One and half hoursafter endotoxin exposure, blood was harvested for TNF measurement.

Blood was centrifuged at 5,000 rpm (1,500×g) for 15 min and thesupernatants were collected for TNF determination. Serum andsupernatants were used for TNF protein analysis by ELISA (R&D SystemsInc., Minneapolis, Minn.) according to the manufacturer's instructions.As shown in FIG. 6, administration of galantamine at 1, 2 and 4 mg/kgsignificantly Inhibited serum TNF in endotoxemia mice.

Results, presented in FIG. 6, indicate that galantamine inhibits serumTNF in endotoxemia mice in a doe-dependent manner. Thus, nearly two-foldinhibition was observed at 2 mg/kg and nearly three-fold inhibition at 4mg/kg of galantamine.

EXAMPLE 8 Galantamine Increases Survival in Murine Model of LethalEndotoxemia

Murine endotoxemia was induced as discussed above in Example 7,Galantamine runs administered intraperitoneally at the doses indicatedin FIG. 7A to 7C (0, 1, 2 or 4 mg/kg) one hour before endotoxinadministration. Mortality was monitored twice daily for 14 days.

FIG. 7A to 7C present the results of three experiments. Thus, 14 daysafter endotoxin administration, 4 mg/kg galantamine increased survivalby about 90% compared with vehicle control. As demonstrated in FIGS. 7Band 7C, on day 14, 1 and 2 mg/kg galantamine increased survival ofendotoxemia rats by about 15 and 25%, respectively.

These results indicate that pre-treatment with galantamine improvessurvival in endotoxemia mice.

EXAMPLE 9 Twine Increases Survival in a Murine Model of Sepsis

Cecal ligation and puncture were performed as described above. Randomlyselected mice were treated intraperitoneally with vehicle (saline, 200ul) or tacrine (2.5 μg/kg or 0.25 μg/kg) twice daily for 3 days,starting 24 hours after surgery. Mice were monitored for survival twicedaily for three weeks. In another set of experiments, mice were injectedintraperitoneally with saline or tacrine (2.5 μg/kg or 0.25 μg/kg) 24hours after surgery and euthanized 6 hours later (30 hours aftersurgery). Blood was obtained by cardiac puncture and prepared for TNFand HMGB1 analyses, TNF was measured by ELISA as discussed above. HMGB1was measured in sera by Western blot as discussed above.

As shown in FIG. 8A, 2.5 μg/kg tacrine, initiated 24 hours after CLP,significantly improved survival in mice with polymicrobial sepsis.Furthermore, as shown in FIGS. 8B and 8C, 2.5 μg/kg tacrinesignificantly reduced scram TNF and HMGB1, respectively.

These results indicate that post-operative treatment with tacrineimproves survival in septic mice and alleviates the symptoms ofinflammation.

EXAMPLE 10 Galantamine Prevents Mortality in Mice with LethalEndotoxemia when Administered 6 Hours Before Endotoxin

Murine endotoxemia was Induced as discussed above in Example 7,Galantamine hydrobromide (4 mg/kg) or vehicle (saline) was administeredintraperitoneally six hours before endotoxin administration. Mortalitywas monitored twice daily for 14 days.

As shown in FIG. 9, 14 days after endotoxin administration, about 75% ofmice treated with galantamine survived whereas none of the animalstreated with vehicle control survived. These results indicate thatpre-treatment with galantamine 6 hours before endotoxin markedlyimproves survival in endotoxemia mice.

EXAMPLE 11 Huperzine A Protects Against Lethal Endotoxemia in Mice

Murine endotoxemia was induced as discussed above in Example 7.Huperzine A (Sigma Chemical Co., St. Louis, Mo.) at 0.4 mg/kg or 0.1mg/kg or vehicle (saline containing the corresponding percent ofmethanol) was administered, intraperitoneally one hour before endotoxinadministration. Mortality was monitored twice daily for 14 days.

As shown in FIG. 10, 14 days after endotoxin administration, about 75%of mice treated with 0.4 mg/kg Huperzine A survived whereas none of theanimals treated with vehicle control survived. These results indicatethat pre-treatment with Huperzine A provides protection against lethalendotoxemia.

EXAMPLE 12 Neostigmine Protects Against Lethal Endotoxemia in Mice

Murine endotoxemia was induced as discussed above in Example 7.Neostigmine methyl sulfate (Sigma Chemical Co., St. Louis, Mo.) at 0.1mg/kg or vehicle (saline) was administered intraperitoneally 30 minutesbefore endotoxin administration. Mortality was monitored twice daily for14 days.

As shown in FIG. 11, 14 days after endotoxin administration, about 50%of mice treated with Neostigmine survived whereas none of the animalstreated with vehicle control survived. These results indicate thatpre-treatment with Neostigmine provides protection against lethalendotoxemia.

EXAMPLE 13 Physostigmine Protects Against Lethal Endotoxemia in Mice

Murine endotoxemia was induced as discussed above in Example 7.Physostigmine (Sigma Chemical Co., St. Louis, Mo.) at 0.2 mg/kg orvehicle (saline) was administered intraperitoneally 30 minutes beforeendotoxin administration. Mortality was monitored twice daily for 14days.

As shown in FIG. 12, 14 days after endotoxin administration, about 80%of mice treated with Physostigmine survived whereas only about 5% of theanimals treated with vehicle control survived. These results Indicatethat pre-treatment with physostigmine provides protection against lethalendotoxemia.

While this Invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a subject with a cytokine-mediated inflammatorydisorder, comprising: administering to the subject a pharmaceuticallyacceptable cholinesterase inhibitor in an amount sufficient to reducethe level of a proinflammatory cytokine, provided that the inhibitor isnot galantamine, wherein the inflammatory disorder is asthma. 2-15.(canceled)
 16. The method of claim 1, wherein the cholinesteraseinhibitor is at least one selected from the group consisting of tacrine,a tacrine analog, fasciculin, metrifonate, heptyl-physostigmine,norpyridostigmine, nomeostigmine, huperzine A or an analogue thereof,physostigmine, heptylphysostigmine, velnacrine, citicoline, donepezil,7-methoxytacrine, eptastigmine, icopezil, ipidacrine, zifrosilone,anseculin, suronacrine, linopiridine, rivastigmine, neostigmine,edrophonium, deinacarium, ambenonium, arecoline, xanomeline,subcomeline, cevimeline, alvameline, milameline, talsaclidine, andcompounds of formulae (XVIII)-(XXI):


17. (canceled)
 18. The method of claim 1 wherein the cholinesteraseinhibitor is at least 95% pure by weight.
 19. The method of claim 16wherein the cholinesterase inhibitor is tacrine.
 20. The method of claim16 wherein the cholinesterase inhibitor is huperzine A.
 21. The methodof claim 16 wherein the cholinesterase inhibitor is neostigmine.
 22. Themethod of claim 16 wherein the cholinesterase inhibitor isphysostigmine.
 23. A method of reducing proinflammatory cytokine levelsin a subject in need of such reduction, comprising administering to thesubject an effective amount of a pharmaceutically acceptablecholinesterase inhibitor, provided that the inhibitor is notgalantamine, wherein the subject suffers from asthma.
 24. A method oftreating a subject suffering from a disorder, comprising: administeringto the subject a therapeutically effective amount of pharmaceuticallyacceptable cholinesterase inhibitor, wherein the disorder is asthma,provided that the inhibitor is not galantamine.